Helicases are a ubiquitous and highly diverse group of enzymes that separate the strands of nucleic acids and are found in bacteria, eukaryotes, archaea, and many viruses. They are essential components of the genome maintenance machinery. Their importance is highlighted in the many human disorders associated with defective helicase function. Many helicases have been shown to carry out multiple, distinct functions in the cell. Often, these processes place very different requirements on the helicase; for instance, one helicase may be tasked with unwinding for short distances, long distances, or not at all, depending on context. How these different functions are defined and regulated remains poorly understood. This project will focus on two proteins, UvrD and XPD, which serve as models for DNA repair helicases in prokaryotes and eukaryotes, respectively. Although they are primarily involved in DNA repair pathways, both helicases also participate in other cellular processes. UvrD and XPD are also prototypes for the two largest structural classes of helicases known, and insights gained on their mechanisms are likely to extend to a number of homologous systems. Prior studies have shown that helicase activity is strongly influenced by oligomeric and conformational state. A monomer can exhibit low or no unwinding activity, but multiple molecules unwind processively; helicases can unwind duplexes in one conformation but displace DNA-bound proteins in another. Helicase roles have thus been proposed to be defined in the cell by protein partners controlling their oligomeric and/or conformational state. These models remain speculative or have not been quantified adequately. In this project, we will investigate the mechanisms by which helicase activity is regulated; first by understanding the factors that limit activity in helicase monomers (Aim 1), next by measuring helicase oligomerization and quantifying how it enhances unwinding activity (Aim 2), and lastly by studying helicase unwinding together with selected protein partners to determine if they exploit the above strategies to regulate helicase activity (Aim 3). These aims will be achieved using a synthesis of single-molecule biophysical techniques?optical tweezers, fluorescence microscopy, and microfluidics?together with traditional biochemical methods. These novel approaches, which exploit the PIs' expertise, will be used to detect the unwinding of helicases at the single molecule level, in real time, and at high resolution, while simultaneously measuring their oligomeric and conformational state. Moreover, these techniques will enable the controlled assembly of multi-component complexes. Beyond providing insights on helicase mechanism and the DNA repair pathways in which they participate, our studies will advance biophysical methods for investigating the dynamics of biomolecular complexes.